The current invention is in the field of protein expression. Herein reported is a promotor with at least one point mutation and a method that uses this promoter to produce a polypeptide.
The expression of proteins is a fundamental process in living cells. All information required for protein expression is provided by a single nucleic acid. This nucleic acid not only contains the information of the protein's amino acid sequence, it also provides the regulatory information required (e.g. the ribosomal binding site, the start and end-signals for transcription, splice signals, enhancer elements, etc.) including a promoter/promoter sequence.
A promoter is a nucleic acid that regulates the amount of transcription of a nucleic acid, e.g. encoding a polypeptide, to which it is operably linked, into pre-mRNA. It is a transcription control element, which is located around the RNA polymerase initiation site at the 5′-end of an operably linked coding sequence. From analysis of the SV40 early promoter it is known that recognition/binding sites for transcription activators are contained in promoters in segments consisting of 7-20 basepairs. One segment is the start site for RNA synthesis, e.g. the well known TATA-box. Other segments, located approximately 30-110 basepairs 5′, i.e. upstream, to the start site for RNA synthesis, are defining the frequency of transcription initiation. A promoter at least requires one segment that initiates RNA synthesis at a specific site and in a defined direction, i.e. in 5′ to 3′ direction.
The gradual loss of productivity in long-term culture is a common issue with the development of manufacturing cell lines (Barnes, L. M., et al. Biotechnol. Bioeng. 81 (2003) 631-639). The decrease of recombinant protein expression can be due to a loss of transgene copies and/or silencing of the transgene promoter (see e.g. Escher, G., et al. J. Lipid Res. 46 (2005) 356-365; Krishnan, M., et al., FASEB J. 20 (2006) 106-108; Yang, Y., et al., J. Biotechnol. 147 (2010) 180-185). Silencing of promoters is caused by epigenetic modifications of chromatin such as posttranslational modifications of histones as well as direct methylation of promoter DNA at CpG sites (see e.g. Cedar, H. and Bergman, Y., Nat. Rev. Genet. 10 (2009) 295-304; De Carvalho, D. D. et al., Trends Cell Biol 20 (2010): 609-617; Klose; R. J. and Bird, A. P., Trends Biochem Sci 31 (2006): 89-97). Methylated promoters are in general inactive.
The very strong promoter and enhancer of the major immediate-early genes of the human cytomegalovirus (hCMV-MIE) is used for the recombinant expression of polypeptides in mammalian cells. It has been shown that the promoter is prone to silencing by methylation in transient as well as stably transfected mammalian cells (see e.g. Escher, G., et al. J. Lipid Res. 46 (2005) 356-365; Krishnan, M., et al., FASEB J. 20 (2006) 106-108; Proesch, S. et al., Biol Chem Heppe Seyler 377 (1996): 195-201; Yang, Y., et al., J. Biotechnol. 147 (2010) 180-185).
It has been demonstrated previously in WO 2011/128377 that direct methylation of the hCMV-MIE promoter can be used as early marker to predict production instability of recombinant CHO cell lines.
Osterlehner, A., et al., report promoter methylation and trans gene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines and describe that the different CpG sites are methylated with different frequency (Biotechnol. Bioeng. 108 (2011) 2670-2681).